Expression cassette and host cell for expressing a Vip3-interacting protein

ABSTRACT

This invention provides polypeptides that were identified as interacting with Vip3 toxin. This invention also provides a method of identifying agents that bind to Vip3 interacting polypeptides, which agents may act as insecticidal agent, cytotoxic agents and/or modulate the activity of Vip3 interacting polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/100,824, filed Aug. 10, 2018, now U.S. Pat. No. 10,836,801, which isa divisional of U.S. application Ser. No. 15/120,563, filed Aug. 22,2016, now abandoned, which is the National Stage of InternationalApplication No. PCT/EP2015/054100, filed Feb. 26, 2015, which claimspriority to U.S. Provisional Application No. 61/945,454, filed Feb. 27,2014, all of which are incorporated herein by reference in theirentirety.

REFERENCE TO A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled “73666-US-REG-D-NAT-2_SeqList_ST25.txt”, 30 kilobytes insin, generated on Sep. 30, 2020 and filed via EFS-Web, is provided inlieu of a paper copy. This Sequence Listing is hereby incorporated byreference into the specification for its disclosures.

FIELD OF THE INVENTION

The present invention relates generally to the field of molecularbiology. More specifically, the field of the invention relates topolypeptides that interact with Vip3 toxin. The polypeptides are usefulfor developing new insecticidal agents.

BACKGROUND

Plant pests are a major factor in the loss of the world's importantagricultural crops. About $8 billion are lost every year in the U.S.alone due to infestations of non-mammalian pests including insects. Inaddition to losses in field crops, insect pests are also a burden tovegetable and fruit growers, to producers of ornamental flowers, and tohome gardeners.

Insect pests are mainly controlled by intensive applications of chemicalpesticides, which are active through inhibition of insect growth,prevention of insect feeding or reproduction, or cause death. Goodinsect control can thus be reached, but these chemicals can sometimesalso affect other, beneficial insects. Another problem resulting fromthe wide use of chemical pesticides is the appearance of resistantinsect varieties. This has been partially alleviated by variousresistance management practices, but there is an increasing need foralternative pest control agents. Biological pest control agents, such asBacillus thuringiensis (Bt) strains expressing pesticidal toxins likeδ-endotoxins, have also been applied to crop plants with satisfactoryresults, offering an alternative or complement to chemical pesticides.The genes coding for some of these δ-endotoxins have been isolated andtheir expression in heterologous hosts have been shown to provideanother tool for the control of economically important insect pests. Inparticular, the expression of Bt δ-endotoxins has provided efficientprotection against selected insect pests, and transgenic plantsexpressing such toxins have been commercialized, allowing farmers toreduce applications of chemical insect control agents.

Another family of insecticidal proteins produced by Bacillus speciesduring the vegetative stage of growth (vegetative insecticidal proteins(Vip)) has also been identified. U.S. Pat. Nos. 5,877,012, 6,107,279,and 6,137,033, herein incorporated by reference, describe a class ofinsecticidal proteins called Vip3. Other disclosures, including WO98/18932, WO 98/33991, WO 98/00546, and WO 99/57282, have also nowidentified homologues of the Vip3 class of proteins. Vip3 codingsequences encode approximately 88 kDa proteins that possess insecticidalactivity against a wide spectrum of lepidopteran pests, including butnot limited to black cutworm (BCW, Agrotis ipsilon), fall armyworm (FAW,Spodoptera frugiperda), tobacco budworm (TBW, Heliothis virescens),sugarcane borer, (SCB, Diatraea saccharalis), lesser cornstalk borer(LCB, Elasmopalpus lignosellus), and corn earworm (CEW, Helicoverpazea). When expressed in transgenic plants, for example corn Zea mays),Vip3 coding sequences confer protection to the plant from insect feedingdamage.

Vip3A is successful as an insecticidal protein in transgenic maize (U.S.Pat. Nos. 6,107,279, 8,232,456 herein incorporated by reference). Theidentification of proteins which interact with a Vip3 provides theability to screen for other agents (e.g., proteins and/or chemicals)that may also have insecticidal properties. The present inventionprovides Vip3 interacting polypeptides and methods for screening forVip3 interacting agents having cytotoxic and/or insecticidal properties.

SUMMARY OF THE INVENTION

The present invention is drawn to polypeptides that interact with Vip3(e.g., from insect pests susceptible to Vip3 toxin) and methods of usingthose polypeptides to identify agents (e.g., ligands) that bind to Vip3interacting polypeptides and/or modulate the activity of at least oneVip3 interacting polypeptide.

The invention includes polypeptides identified as interacting with Vip3.In representative embodiments, the invention provides recombinant Vip3interacting polypeptides. In embodiments, the invention providesnon-naturally occurring Vip3 interacting polypeptides. Optionally theVip3 interacting polypeptide comprises, consists essentially of, orconsists of the amino acid sequence of any of SEQ ID NOs: 1-8 or asubstantially similar or substantially identical amino acid sequencethereto.

The invention also includes recombinant polynucleotide sequences whichencode the above polypeptides, and vectors, expression cassettes andcells comprising the above mentioned polynucleotide sequences.

The invention includes a method for using Vip3-interacting polypeptidesto identify other polypeptides that act in the same pathway, therebyelucidating mode of action; to identify agents that interact with (e.g.,bind) Vip3-interacting polypeptides, to identify agents that haveinsecticidal properties; to identify polypeptides that bind to Vip3, andto elucidate the mode of action of a different Vip3 in a differentinsect system.

Also provided by the invention is a method of identifying an agent thatinteracts with at least one Vip3 interacting polypeptide. The agent mayinteract with a single Vip3 interacting polypeptide, with multiple Vip3interacting polypeptides individually, or with a complex of Vip3interacting polypeptides which may include other polypeptides (that door do not interact with Vip3). In a representative embodiment, themethod of identifying the agent comprises contacting at least one Vip3interacting polypeptide or fragment thereof (optionally, a biologicallyactive fragment), acting individually or as part of a complex, to one ormore test agents, and then detecting binding activity between the Vip3interacting polypeptide(s) under conditions sufficient for binding.Binding activity can be measured by any method known in the art, e.g.,by measuring desired binding characteristics, including but not limitedto binding affinity, binding site specificity, and/or association anddissociation rates. Binding activity can also be determinedqualitatively, such as in a gel-shift assay. In representativeembodiments, binding between the Vip3 interacting polypeptide(s) and thetest agent indicates that the test agent is a candidate insecticidalagent.

In another embodiment of the invention, the method further comprisesmeasuring the cytotoxicity and/or insecticidal activity of the candidateinsecticidal agent (e.g., in a cell based or live insect based system).

Also provided by the invention is a method of identifying an agent thatmodulates the activity of at least one Vip3 interacting polypeptide. Inrepresentative embodiments, the method comprises contacting at least oneVip3 interacting polypeptide or a fragment thereof (optionally, abiologically active fragment) with a test agent in a cell (e.g., aninsect cell), for example, from a heterologous nucleotide sequence(s),and detecting cytotoxicity under conditions sufficient to providecytotoxicity. A change in the level of cytotoxicity compared to thelevel of cytotoxicity in the absence of the test agent indicates thatthe test agent is an agent that modulates the activity of at least oneVip3 interacting polypeptide.

Optionally, in fixe methods of the invention, the agent is a Bacilluspolypeptide(s).

Also provided by the invention is a method of identifying aninsecticidal and/or cytotoxic agent that interacts with at least oneVip3 interacting polypeptide or a fragment (e.g., a biologically activefragment) thereof. In representative embodiments, the method comprisescontacting at least one Vip3 interacting polypeptide or fragment thereofto a Bacillus polypeptide(s); isolating a Vip3 interacting polypeptideunder conditions appropriate for co-purification with a ligand,identifying a co-purified polypeptide, and determining the insecticidaland/or cytotoxic activity of the co-purified polypeptide.

Further provided is a method of identifying a cytotoxic agent thatinteracts with at least one Vip3 interacting polypeptide, said methodcomprising: a. providing at least one Vip3 interacting polypeptide orfragment thereof (e.g., a biologically active fragment) expressed in acell (optionally an insect cell), wherein the cell is not susceptible toVip3 toxin; b. contacting the at least one Vip3 interacting polypeptideor fragment with a test agent under conditions sufficient to promotebinding, and detecting cytotoxicity of the cell, wherein an increase inthe level cytotoxicity as compared with the level of cytotoxicity in theabsence of the test agent indicates that the test agent is a cytotoxicagent. In embodiments, the cytotoxicity of the combination of the Vip3interacting polypeptide and the test agent is greater (e.g., at least25%, 50%, 75%, 100%, 200% or more greater) than the cytotoxicity ofeither alone. In representative embodiments, neither the Vip3interacting polypeptide nor the test agent shows significantcytotoxicity on its own.

In another embodiment of the invention, test agents can compriseenvironmental samples, biological samples, chemical libraries, orcellular extracts.

A further embodiment is a ligand of a Vip3 interacting polypeptideidentified by a method of the invention.

Another embodiment is a candidate insecticidal agent identified by amethod of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO 1: depicts the polypeptide sequence of ATP synthase α from S.frugiperda.

SEQ ID NO 2: depicts the polypeptide sequence of ATP synthase β from S.frugiperda

SEQ ID NO 3: depicts the polypeptide sequence of Hsc70 from S.frugiperda

SEQ NO 4: depicts the polypeptide sequence of prohibitin-1 from H. zea.

SEQ ID NO 5: depicts the polypeptide sequence of prohibitin-1 from M.sexta

SEQ ID NO 6: depicts the polypeptide sequence of prohibitin-2 from H.zea

SEQ ID NO 7: depicts the polypeptide sequence of prohibitin-2 from M.sexta.

SEQ ID NO 8: depicts the polypeptide sequence of serpin from H. sexta.

SEQ ID NO 9: depicts one strand of the double-stranded RNA of Hsc70 fromS. frugiperda used for dsRNA experiments in examples 12 and 13.

SEQ NO 10: depicts one strand of the double-stranded RNA of ATP synthaseα from S. frugiperda used for dsRNA experiments in examples 12 and 13.

SEQ ID NO 11: depicts one strand of the double-stranded RNA of ATPsynthase β from S. frugiperda used for dsRNA experiments in examples 12and 13.

DETAILED DESCRIPTION OF THE INVENTION

This description is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure, which do not depart from the instant invention.Hence, the following descriptions are intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety

As used herein, “a,” “an” or “the” can mean one or more than one. Forexample, a cell can mean a single cell or a multiplicity of cells.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (or).

Further, the term “about,” as used herein when referring to a measurablevalue such as an amount of a compound or agent, dose, time, temperature,and the like, is meant to encompass variations of =20%, ±10%, +5%, ±1%,+0.5%, or even ±0.1% of the specified amount.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention. Thus, the term “consisting essentially of” when used in aclaim of this invention is not intended to be interpreted to beequivalent to “comprising.”

It will be understood that, although the terms “first,” “second,” etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. Thus, a “first” element (e.g., a firstpromoter sequence) as described herein could also be termed a “second”element (e.g., a second promoter sequence) without departing from theteachings of the present invention.

For purposes of the present invention, “insect” or “insect pest” includewithout limitation insects and arachnids selected from the ordersColeoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera,Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura,Siphonaptera, Trichoptera, and Acari, particularly Coleoptera andLepidoptera. In particular, insect pests include black cutworm (Agrotisipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (S.exigua), yellow striped armyworm (S. ornithogalli), southwestern cornborer (Diatraea grandiosella), sugarcane borer (D. saccharalis), cornearworm (Helicoverpa zea), Mediterranean corn borer (Sesamianonagroides), cabbage looper (Trichoplusia ni), velvetbean caterpillar(Anticarsia gemmatalis), diamondback moth (Plutella xylostella), tobaccobudworm (Heliothis virescens), European corn borer (Ostrinia nubilalis),western corn rootworm (Diabrotica virgifera virgifera), southern cornrootworm (Diabrotica undecimpunctata howardi), northern corn rootworm(Diabrotica barberi).

The term “Vip3” as used herein is intended broadly and encompassesnaturally occurring Vip3 polypeptides including but is not limited toVip3A (e.g., Vip3A(a), Vip3A(b), Vip3A(c)), Vip3B, Vip3C, Vip3D, Vip3E)and any other protein now known or later identified as a Vip3 (see.e.g., the database of Vip3 toxin nomenclature found on the world-wideweb at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html) andequivalents of any of the foregoing (including engineered Vip3polypeptides and Vip3 fragments). The term Vip3 also includespolypeptides that are substantially similar or substantially identicalat the amino acid level to the toxic core region of a Vip3 protein,optionally while also substantially retaining at least one Vip3biological activity (e.g., insecticidal and/or cytotoxic activity). Theterm “Vip3” further includes modifications (e.g., deletions and/ortruncations) of a naturally occurring Vip3 or an equivalent thereof thathas a substantially similar or substantially identical amino acidsequence to a naturally occurring Vip3. Exemplary Vip3 equivalents havebeen disclosed in 8118932, WO 98/3399_, WO 98/00546, and WO 99/57282.

Further, the Vip3 can be from any bacterial genus or species of originincluding without limitation a Bacillus species e.g., B. cerues, B.thuringiensis, and the like), Clostridium, or other soil-borne bacteria.

In embodiments, a Vip3 equivalent comprises a fragment of anaturally-occurring or non-naturally occurring full-length Vip3polypeptide, optionally a biologically active fragment.

In representative embodiments, a biologically active equivalent of aVip3 polypeptide, a biologically active fragment of a Vip3 polypeptide,or a biologically active equivalent thereof, comprises the toxic coreregion of a naturally-occurring Vip3 protein, or an amino acid sequencethat is substantially similar or substantially identical to the toxiccore region of a naturally-occurring Vip3 protein.

A polypeptide that is “substantially similar” or “substantiallyidentical” to a reference amino acid sequence is 1) a polypeptide thatis at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, ormore than 99% similar or identical at the amino acid sequence level tothe referenced polypeptide, optionally while also substantiallyretaining at least one activity associated with the referencepolypeptide, 2) a polypeptide that is cross-reactive to an antibody thatimmunologically recognizes the reference polypeptide, and/or 3) apolypeptide that is cross-reactive with a receptor bound by thereferenced polypeptide and, optionally, acts as a receptor agonist.

The proteins of the Vip3 class are secreted into the media by Bacillusspp, in vegetative stages of growth. For example, the Vip3: protein(e.g., Vip3A(a) protein) is a member of a distinct class of proteinsdisplaying insecticidal activity against a broad spectrum oflepidopteran insects including black cutworm (Agrotis ipsilon), fallarmyworm (Spodoptera frugiperda), tobacco hornworm (Manduca sexta), beetarmyworm (S. exigua), yellow striped armyworm (S. ornithogalli),southwestern corn borer (Diatraea grandiosella), sugarcane borer (D.saccharalis), corn earworm (Helicoverpa zea), Mediterranean corn borer(Sesamia nonagroides), cabbage looper (Trichoplusia ni), velvetbeancaterpillar (Anticarsia gemmatalis), diamondback moth (Plutellaxylostella) and tobacco budworm (Heliothis virescens).

As discussed above, Vip3A protein (e.g., Vip3A(a) protein) has beenshown to be active against a broad spectrum of plant pests. For example,histopathological observations indicate that Vip3A ingestion bysusceptible insects such as black cutworm (Agrotis epsilon) and fallarmyworm (Spodoptera frugiperda) causes gut paralysis at concentrationsas low as 4 ng/cm² of diet, with complete lysis of the gut epithelialcells resulting in larval death at concentrations above 40 ng/cm². Lesssusceptible insects like European corn borer (Ostrinia nubilalis) do notdevelop any pathology upon ingesting Vip3A. While the proteolyticprocessing of the Vip3A protein by midgut fluids obtained fromsusceptible and non-susceptible insects is comparable, in vivoimmuno-localization studies show that Vip3A(a) binding is restricted togut cells of susceptible insects. Therefore, the insect host range forVip3A seems to be determined by its binding ability to gut cells.Histopathological observations indicate that midgut epithelial cells ofsusceptible insects are the primary target for the Vip3A insecticidalprotein and their subsequent lysis is the primary mechanism oflethality.

Immunohistochemistry indicates that Vip3A (e.g., Vip3A(a)) has theability to bind to the apical membranes of midgut epithelial cells andthat this binding triggers the process that will eventually end withcell lysis. This indicates that there exists one or more proteinslocated in the apical membrane that recognize and bind to Vip3A.

As used herein, the “activity” or “biological activity” of a Vip3protein includes any biological activity of a Vip3 protein includingwithout limitation insecticidal activity, cytotoxic activity and/orbinding activity.

“Insecticidal activity” refers to activity as an insect control agent(e.g., an orally active insect control agent), for example, byinhibiting, through a toxic effect, the ability of one or more insectspecies to survive, grow, feed, and/or reproduce, which may or may notcause death of the insect.

As used herein, to “control insects” (and similar terms) means toinhibit, through a toxic effect, the ability of an insect pest tosurvive, grow, feed, and/or reproduce, and/or to limit insect-relateddamage and/or loss in crop plants. The term “control insects” may or maynot mean killing the insects, although in representative embodiments,one or more insect pests are killed.

As used herein, “toxicity” refers to the decreased viability of a cell,and “viability” refers to the ability of a cell to proliferate and/ordifferentiate and/or maintain its biological characteristics in a mannercharacteristic of that cell in the absence of a particular cytotoxicagent.

“Expression cassette” as used herein means a nucleic acid (e.g., DNA)sequence capable of directing expression of a gene in a cell (e.g., aplant, insect, or bacterial cell), comprising a promoter operably linkedto an amino acid coding region which is operably linked to a terminationregion. The gene may be chimeric, meaning that at least one component ofthe gene is heterologous with respect to at least one other component ofthe gene. The gene may also be naturally occurring, but which has beenobtained in a recombinant form useful for genetic transformation of aplant or microorganism.

As used herein, the term “nucleic acid,” “nucleic acid molecule,”“polynucleotide” and/or “nucleotide sequence” refers to a heteropolymerof nucleotides or the sequence of these nucleotides from the 5′ to 3′end of a nucleic acid molecule and includes DNA or RNA molecules,including cDNA, a DNA fragment, genomic DNA, synthetic (e.g.,chemically, synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, anyof which can be single stranded or double stranded or a combination ofboth. The terms “nucleotide sequence” “nucleic acid,” “nucleic acidmolecule,” “oligonucleotide” and “polynucleotide” are also usedinterchangeably herein to refer to a heteropolymer of nucleotides.Nucleic acid sequences provided herein are presented herein in the 5′ to3′ direction, from left to right and are represented using the standardcode for representing the nucleotide characters as set forth in thesequence rules for the U.S. Patent and Trademark Office, 37 CFR §§1.821-1.825, and the World Intellectual Property Organization (WIPO)Standard ST.25.

In embodiments, nucleic acids according to the present invention arenon-naturally occurring nucleic acids. In embodiments, nucleic acidsaccording to the present invention are recombinant nucleic acids.

As used herein, the term “gene” is used broadly to refer to any segmentof nucleic acid associated with a biological function. Thus, genesinclude coding sequences and/or the regulatory sequences required fortheir expression. For example, “gene” refers to a nucleic acid fragmentthat expresses mRNA or functional RNA, or encodes a specific protein,and which includes regulatory sequences. Genes also include nonexpressedDNA segments that, for example, form recognition sequences for otherproteins. Genes can be obtained from a variety of sources, includingcloning from a source of interest or synthesizing from known orpredicted sequence information, and may include sequences designed tohave desired parameters.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” in which onlysome of the nucleotides bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands.

An “isolated” nucleic acid or polynucleotide of the present invention isgenerally free of nucleic acid sequences that flank the nucleic acid ofinterest in the genomic DNA of the organism from which the nucleic acidwas derived (such as coding sequences present at the 5′ or 3′ ends).However, the nucleic acid of this invention can include some additionalbases or moieties that do not deleteriously affect the basic structuraland/or functional characteristics of the nucleic acid. An “isolated”nucleic acid or polynucleotide includes a chimeric molecule or a cDNAwhich may not be naturally occurring. “Isolated” does not necessarilymean that the preparation is technically pure (homogeneous).

Thus, an “isolated nucleic acid” or “isolated polynucleotide” is presentin a form or setting that is different from that in which it is found innature and is not immediately contiguous with nucleotide sequences withwhich it is immediately contiguous (one on the 5′ end and one on the 3′end) in the naturally occurring genome of the organism from which it isderived. Accordingly, in one embodiment, an isolated nucleic acidincludes some or all of the 5′ non-coding (e.g., promoter) sequencesthat are immediately contiguous to a coding sequence. The term thereforeincludes, for example, a recombinant nucleic acid that is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment), independent of othersequences. Thus, a nucleic acid found in nature that is removed from itsnative environment and transformed into a plant is still considered“isolated” even when incorporated into the genome of the resultingtransgenic plant. It also includes a recombinant nucleic acid that ispart of a hybrid nucleic acid encoding an additional polypeptide orpeptide sequence.

The term “isolated” can further refer to a nucleic acid, nucleotidesequence, polypeptide, peptide or fragment that is substantially free ofcellular material, viral material, and/or culture medium (e.g., whenproduced by recombinant DNA techniques), or chemical precursors or otherchemicals (e.g., when chemically synthesized). Moreover, an “isolatedfragment” is a fragment of a nucleic acid, nucleotide sequence orpolypeptide that is not naturally occurring as a fragment and would notbe found as such in the natural state. “Isolated” does not mean that thepreparation is technically pure (homogeneous), hut it is sufficientlypure to provide the polypeptide or nucleic acid in a form in which itcan be used for the intended purpose.

The terms “polypeptide,” “protein,” and “peptide” refer to a chain ofcovalently linked amino acids. In general, the term “peptide” can referto shorter chains of amino acids (e.g., 2-50 amino acids); however, allthree terms overlap with respect to the length of the amino acid chain.As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and encompass peptides, unless indicated otherwise.Polypeptides, proteins, and peptides may comprise naturally occurringamino acids, non-naturally occurring amino acids, or a combination ofboth. The polypeptides, proteins, and peptides may be isolated fromsources (e.g., cells or tissues) which they naturally occur, producedrecombinantly in cells in vivo or in vitro or in a test tube in vitro,or synthesized chemically. Such techniques are known to those skilled inthe art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Ausubel et al. CurrentProtocols in Molecular Biology (Green Publishing Associates, Inc. andJohn Wiley & Sons, Inc., New York).

In embodiments, the polypeptides and polynucleotides of the inventioncan be recombinant (produced by genetic engineering), and can optionallybe chimeric. In representative embodiments, polypeptides according tothe present invention are non-naturally occurring polypeptides.

The term “fragment,” as applied to a polypeptide, will be understood tomean an amino acid sequence of reduced length relative to a referencepolypeptide or amino acid sequence and comprising, consistingessentially of, and/or consisting of an amino acid sequence ofcontiguous amino acids identical to the reference polypeptide or aminoacid sequence. Such a polypeptide fragment according to the inventionmay be, where appropriate, included in a larger polypeptide of which itis a constituent (e.g., a fusion protein). In some embodiments, suchfragments can comprise, consist essentially of, and/or consist ofpeptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25,30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acidsof a polypeptide or amino acid sequence according to the invention. Afragment of a polypeptide or protein can be produced by methods wellknown and routine in the art, for example, by enzymatic or othercleavage of naturally occurring peptides or polypeptides or by syntheticprotocols that are well known.

A polypeptide fragment can optionally be a biologically active fragment.A “biologically active fragment” or “active fragment” refers to afragment that substantially retains (e.g., at least about 25%, 30% 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) oneor more of the biological activities of the reference polypeptide. Suchfragments can be tested for biological activities according to methodsdescribed in the art. For example, an active fragment of a Vip3 proteincan be tested for binding to a Vip3 interacting polypeptide,insecticidal activity, and/or cytotoxic activity. Thus, the presentinvention further provides biologically active fragments of apolypeptide and the polynucleotides encoding such biologically activepolypeptide fragments.

The term “agent” as used herein refers to a molecule that is a candidatebinding partner e.g., ligand) for a Vip3 interacting polypeptide and/orcandidate toxin identified by its cytotoxic effect in the presence of aVip3 interacting polypeptide. The term “ligand” as used herein refers toa substance that can specifically bind with a biomolecule, such as aVip3 interacting polypeptide. Optionally, the agent can modulate theactivity (e.g., an antagonist or an agonist) of its binding partner(e.g., Vip3 interacting polypeptide) and/or a downstream pathway. Inrepresentative embodiments, the “agent” is a polypeptide (includingmodified polypeptides such as glycoproteins), a carbohydrate (includinga sugar), a lipid, a nucleic acid (including nucleic acids comprisingmodified bases or a small molecule. In representative embodiments, thetest agent comprises a protein extract or polypeptide from one or moreBacillus spp. Candidate agents further include molecules available fromdiverse libraries of small molecules created by combinatorial syntheticmethods. Candidate agents also include, but are not limited toantibodies, peptides, aptamers, and other small molecules designed ordeduced to interact with the Vip3 interacting polypeptides of theinvention. Agents identified by the screening methods of the inventioninclude potential novel insecticidal toxins, the insecticidal activityof which can be determined by known methods, such as feeding assays asdescribed in U.S. Pat. Nos. 5,407,454; 6,232,439; and Marrone et al(1985) J. of Economic Entomology 78:290-293 (each herein incorporated byreference).

As used herein, the term “modulate” or “modulates” (and similar terms)indicates an increase or decrease in the referenced activity of at leastabout 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 100%, 200%, or more.

An “environmental sample” is a sample of any material that is collectedfrom an environmental source, including but not limited to water samplessuch as that from ponds, lakes, or rivers; soil samples; samples ofair-borne particles; samples of vegetation; or smear samples fromsurfaces.

A “biological sample” is a sample of any material that is collected froma biological source, including but not limited to tissue samples fromany part of any plant, insect, or animal, including blood, serum, orcell culture; or microorganism cultures, including but not limited tosuch microorganisms as fungi, yeast, bacteria, or algae.

A “chemical library” is a series of stored chemicals. Each chemical hasassociated information stored in some kind of database with informationincluding but not limited to the chemical structure, purity, quantity,and/or physiochemical characteristics of the compound. Chemicallibraries include molecules available from diverse libraries of smallmolecules created by combinatorial synthetic methods.

The present invention is based, in part, on the identification of Vip3interacting polypeptides from insects susceptible to Vip3 toxin based ontheir interaction with Vip3. A “Vip3 interacting polypeptide” is apolypeptide that interacts (e.g., binds covalently and/ornon-covalently) with Vip3, for example, in a binding assay. Inembodiments, the Vip3 interacting polypeptide is from an insectsusceptible to Vip3 toxin, although it may be produced in recombinantform rather than being isolated from its native source. In embodiments,the Vip3 interacting polypeptide binds directly to Vip3. In embodiments,the Vip3 interacting polypeptide binds indirectly to Vip3 (e.g., as partof a complex, another component of which binds directly to Vip3). Inembodiments, the Vip3 interacting polypeptide of the invention is ATPsynthase α, ATP synthase β, Hsc-70, prohibitin-1, prohibitin-2, and/orserpin. These polypeptides may interact with Vip3 individually, witheach other, and/or with other proteins (e.g., insect proteins), and mayoptionally participate in the mod of action of Vip3. Combinationscomprising Vip3 and ATP synthase α, ATP synthase β, prohibitin-1,prohibitin-2, serpin, and/or other proteins (e.g., insect proteins) thatresult in insectidical activity and/or cytotoxicity of insect cells(e.g., insect cells susceptible to Vip3 toxin) are also encompassed inthe present invention.

In representative embodiments, the Vip3 interacting agent is provided aspart of a cellular extract.

In representative embodiments, the agent interacting with Vip3interacting polypeptide is provided is provided as part of a cellularextract.

In representative embodiments, the Vip3 interacting polypeptide of theinvention binds to a Vip3 and/or mediates cytotoxic activity in thepresence of Vip3.

In representative embodiments, the Vip3 interacting polypeptidecomprises, consists essentially of, or consists of prohibitin-1 andprohibitin-2.

In representative embodiments, the Vip3 interacting polypeptidecomprises, consists essentially of, or consists of ATP synthase α andATP synthase β.

The terms “ATP synthase α”, “ATP synthase β”, “Hsc-70”, “prohibitin-1”,“prohibitin-2”, and “serpin” are intended broadly herein and encompassnaturally occurring polypeptides now known or later identified andequivalents of any of the foregoing (including engineered polypeptidesand fragments) that retain at least one biological activity of thenative protein (e.g., binds Vip3 and/or forms a complex that bindsVip3). These terms further include modifications (e.g., deletions and/ortruncations) of a naturally occurring polypeptide or an equivalentthereof that has a substantially similar or substantially identicalamino acid sequence to a naturally occurring polypeptide and thatretains at least one biological activity associated with the naturallyoccurring polypeptide. Further, the ATP synthase α, ATP synthase β,Hsc-70, prohibitin-1, prohibitin-2, or serpin polypeptide can be fromany species of origin including without limitation an insect species(for example, from a lepidopteran insect species, from a coleopteraninsect species, from the genus Spodoptera [e.g., S. frugiperda], thegenus Helicoverpa [e.g., H. zea], the genus Manduca [e.g., M. sexta]),the genus Ostrinia (e.g. O. nubilalis) or the genus Diabrotica (e.g. D.virgifera). Those skilled in the art will appreciate that the specificVip3 interacting polypeptides and nucleic acids encoding the samedisclosed herein can be used to identify corresponding Vip3 interactingpolypeptides in other species (e.g., insect species, optionally a Vip3susceptible insect species).

In embodiments, an ATP synthase α, ATP synthase β, Hsc-70, prohibitin-1,prohibitin-2, or serpin equivalent comprises a fragment of anaturally-occurring or non-naturally occurring full-length polypeptide,optionally a biologically active fragment (e.g., substantially retainsthe ability to interact with Vip3, to bind Vip3, to form a complex thatinteracts with or binds Vip3, enzymatic activity and/or proteaseinhibition activity).

In representative embodiments, a biologically active equivalent of anATP synthase α, ATP synthase β, Hsc-70, prohibitin-1, prohibitin-2, orserpin polypeptide or a biologically active fragment thereof is asoluble fragment that comprises a Vip3 binding domain, or an amino acidsequence that is substantially similar or substantially identical to theVip3 binding domain of a naturally-occurring ATP synthase α, ATPsynthase β, Hsc-70, prohibitin-1, prohibitin-2, and/or serpin.

In representative embodiments, the Vip3 interacting polypeptidecomprises, consists essentially of, or consists of ATP synthase α fromSpodoptera frugiperda, ATP synthase β from S. frugiperda, Hsc70 from S.frugiperda, prohibitin-1 from Helicoverpa zea and/or from Manduca sexta,prohibitin-2 from H. zea and/or from M. sexta, or serpin from Manducasexta, and any combination of the foregoing.

In representative embodiments, the Vip3 interacting polypeptidecomprises, consists essentially of, or consists of the amino acidsequence of any of SEQ ID NOS: 1-8 or equivalents thereof (includingfragments and equivalents thereof). Equivalents of the Vip3 interactingpolypeptides encompass those that have substantial amino acid sequenceidentity or similarity with any of SEQ ID NOS: 1-8 or a fragmentthereof, optionally a biologically active fragment.

It will be understood that naturally occurring polypeptides willtypically tolerate substitutions in the amino acid sequence andsubstantially retain biological activity. To routinely identifybiologically active polypeptides other than naturally occurring Vip3 orthe Vip3 interacting polypeptides of SEQ ID NOs: 1-8), amino acidsubstitutions may be based on any characteristic known in the art,including the relative similarity or differences of the amino acidside-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. In particular embodiments,conservative substitutions (i.e., substitution with an amino acidresidue having similar properties) are made in the amino acid sequenceencoding the Vip3 polypeptide or the Vip3 interacting polypeptide.

The invention further provides polynucleotides encoding the Vip3interacting proteins of the invention, and vectors, expression cassettesand host cells comprising the same.

The compositions of the present invention are useful for, among otheruses, expressing the Vip3 interacting polypeptides in cells to producecellular or isolated preparations of the polypeptides for investigatingthe structure-function relationships of the Vip3 interactingpolypeptides; investigating their interactions with Vip3 toxin;elucidating the mode of action n of Vip3 toxins; screening andidentifying agents as ligands of Vip3 interacting polypeptides includinginsecticidal or cytotoxic toxins; and designing and developing ligandsof Vip3 interacting polypeptides including insecticidal toxins.

Screening for ligands of Vip3 interacting polypeptides can be performedin a number of ways. For example, at least one recombinant or isolatednucleotide sequence encoding a Vip3 interacting polypeptide of theinvention can be expressed in a cell of interest, and utilized in intactcell or in-vitro receptor binding assays, and/or intact cell toxicityassays. Methods and conditions for insect toxin binding and toxicityassays are known in the art and include but are not limited to thosedescribed in U.S. Pat. No. 5,693,491; Keeton et al. (1998) Appl.Environ. Microbiol. 64(6):2158-2165; Francis et al. (1997) InsectBiochem. Mol. Bio. 27(6):541-550; Keeton et al, (1997) Appl. Environ.Microbiol. 63(9):3419-3425; Vadlamudi et al. (1995) J. Biol. Chem.270(10):5490-5494; Ihara et al. (1998) Comparative Biochem. Physiol. B25120:197-204; and Nagamatsu et al. (1998) Biosci. Biotechnol, each hereinincorporated by reference.

The term “cell of interest” as used herein refers to any cell in whichexpression of the polypeptides of the invention is desired. Cells ofinterest include, but are not limited to mammalian, avian, insect,plant, bacteria, fungi and yeast cells. Cells of interest furtherinclude but are not limited to cultured cell lines, primary cellcultures, cells in vivo, and cells of transgenic organisms.

The methods of the invention encompass using the polypeptides encoded bythe nucleotide sequences of the invention in binding and/or toxicityassays to identify agents (e.g., ligands) that interact with a Vip3toxin interacting polypeptide, including agents that are agonists orantagonists.

Agents may act by modulating the activity of a Vip3 interactingpolypeptide, which, for example, may change the level of cytotoxicity ascompared to the level of cytotoxicity in the absence of the agent.Modulating the activity may result in either an increase or decrease ofcytotoxicity compared to the absence of the agent.

The invention provides methods for screening agents (e.g., ligands) thatbind directly or indirectly to the Vip3 interacting polypeptidesdescribed herein. Both the Vip3 interacting polypeptides and fragmentsthereof (for example, the Vip3 toxin binding domain) can be used toscreen for compounds that bind to at least one of the Vip3 interactingpolypeptides and fragments thereof and optionally exhibit desiredbinding characteristics. Desired binding characteristics include but arenot limited to binding affinity, binding site specificity, associationand dissociation rates, and the like. The screening assay can be anintact cell or in vitro assay that includes exposing a Vip3 toxinbinding domain to a sample ligand or agent and detecting the formationof a ligand-binding polypeptide complex. The assay can be a directligand-polypeptide binding assay or a ligand competition assay.

In representative embodiments, the Vip3 interacting polypeptide is afusion protein comprising a detectable moiety (e.g., a poly-histidinesequence, a FLAG epitope, glutathione-S-transferase, maltose-bindingprotein, or a reporter protein [for example, Green Fluorescent Protein,ß-glucuronidase, ß-galactosidase, luciferase, etc.], hemagglutinin,c-myc, and the like.

For in vitro binding assays, the Vip3 interacting polypeptide(s) may beprovided as isolated, lysed cellular extracts, or homogenized cellularpreparations or a recombinant polypeptide. The polypeptides may beprovided in solution, or immobilized to a matrix. Methods forimmobilizing polypeptides are well known in the art, and include but arenot limited to construction and use of fusion polypeptides withcommercially available high affinity ligands. For example, GST fusionproteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtiter plates.The polypeptides can also be immobilized utilizing techniques in theart, such as using the conjugation of biotin and streptavidin. Thepolypeptides can also be immobilized utilizing techniques in the artutilizing chemical conjugation (linking) of polypeptides to a matrix.Alternatively, the polypeptides may be provided in intact cell bindingassays in which the polypeptides are generally expressed fromrecombinant nucleic acids and the interaction is detected in situ usingtechniques in the art, such as FRET (see for example Masi et al. (2010)Adv. Exp. Ailed. Biol. 674: 33-42 and references cited within).

The present invention may utilize intact cell toxicity assays to screenfor agents that hind to the Vip3 interacting polypeptides describedherein and confer toxicity upon a cell of interest expressing thepolypeptide. An agent selected by this screening method is a candidate ainsecticidal agent (e.g., for an insect expressing the Vip3 interactingpolypeptide), particularly enterally. Toxicity assays include withoutlimitation exposing, in intact cells expressing a polypeptide of theinvention, the toxin binding domain of the polypeptide to a sample agentand detecting the toxicity effected in the cell expressing thepolypeptide.

In one embodiment, the methods of the present invention compriseproviding at least Vip3 interacting polypeptide of the invention,contacting the polypeptide with a test agent under conditions promotingbinding, and determining the viability of the cell expressing the cellsurface Vip3 toxin interacting polypeptide. As used herein, “contacting”(and similar terms) means that the test agents are presented to theintended ligand binding site of the polypeptides of the invention.“Conditions promoting binding” (and similar terms) refers to anycombination of physical and biochemical conditions that enables a ligandof the polypeptides of the invention to determinably bind the intendedpolypeptide over background levels. Examples of such conditions forbinding of ligands to insect toxin receptors, as well as methods forassessing the binding, are known in the art and include but are notlimited to those described in Keeton et al. (1998) Appl EnvironMicrobiol 64(6): 2158-2165; Francis et al. (1997) Insect Biochem MolBiol 27(6):541-550; Keeton et al. (1997) Appl Environ Microbiol63(9):3419-3425; Vadlamudi et al. (1995) J Biol Chem 270(10):5490-5494;Ihara et al. (1998) Comparative Biochemistry and Physiology, Part B120:197-204; and Nagamatsu et al. (1998) Biosci. Biotechnol. Biochem.62(4):727-734, the contents of each which are herein incorporated byreference.

In carrying out the present invention, commercially available methodsfor studying protein-protein interactions, such as yeast and/orbacterial two-hybrid systems can also be used. Two-hybrid systems areavailable from, for example, CLONTECH (Palo Alto, Calif.).

The compositions and screening methods of the invention are useful fordesigning and developing ligands of Vip3 interacting polypeptidesincluding insecticidal toxins. Candidate agents screened andcharacterized for binding, toxicity, and/or species specificity and/orligands having known characteristics and specificities can be linkedand/or modified to produce ligands having particularly desiredcharacteristics and specificities. The methods described herein forassessing binding, toxicity and insecticidal activity can be used toscreen and characterize the ligands.

A variety of sources can be used for screening for other agents thatinteract with the identified Vip3 interacting proteins. This includesbut is not limited to biological samples, environmental samples,cellular extracts, or extracts from microorganisms such as Bacillusspecies. The cellular extracts tested in the methods of the presentinvention can be, as examples, aqueous extracts of cells or tissues,organic extracts of cells or tissues or partially purified cellularfractions. Extracts may comprise intracellular proteins, extracellularproteins, total cell extracts, or at least partially purifiedsubcellular fractions. A skilled artisan can readily recognize thatthere is no limit as to the source of the cellular extract used in thescreening method of the present invention.

Another embodiment of the present invention provides methods foridentifying agents that bind to a Vip3 interacting polypeptide. In anexemplary embodiment, agents that bind to a Vip3 interacting polypeptidecan be identified by: 1) the ability of the agent to bind at least oneVip3 interacting polypeptide, 2) the ability to block Vip3 binding to aVip3 interacting polypeptide, and/or 3) the ability to kill cellsexpressing a Vip3 interacting polypeptide.

More particularly, in one embodiment, at least one Vip3 interactingpolypeptide is mixed with an agent (e.g., a cellular extract or isolatedmolecule(s)). After mixing under conditions that allow association ofthe Vip3 interacting polypeptide(s) with the agent, the mixture isanalyzed to determine if the agent binds to the Vip3 interactingpolypeptide(s).

As another alternative, targets that are bound by a Vip3 interactingpolypeptide can be identified using a yeast two-hybrid system or using abinding-capture assay. In the yeast two hybrid system, an expressionunit encoding a fusion protein made up of one subunit of a two subunittranscription factor and the Vip3 interacting polypeptide is introducedand expressed in a yeast cell. The cell is further modified tocontain 1) an expression unit encoding a detectable marker whoseexpression requires the two subunit transcription factor for expressionand 2) an expression unit that encodes a fusion protein made up of thesecond subunit of the transcription factor and a cloned segment of DNA.If the cloned segment of DNA encodes a protein that binds to the Vip3interacting polypeptide, the expression results in the interaction ofthe Vip3 interacting polypeptide and the encoded protein. This bringsthe two subunits of the transcription factor into binding proximity,allowing reconstitution of the transcription factor. This results in theexpression of the detectable marker. The yeast two hybrid system isparticularly useful in screening a library of cDNA encoding segments forcellular binding partners of the Vip3 interacting polypeptide.

The Vip3 interacting polypeptide used in the above assays can be arecombinant or isolated polypeptide, a fragment of a Vip3 interactingpolypeptide (such as a soluble fragment containing the Vip3 bindingsite), a cell that has been altered to express a Vip3 interactingpolypeptide or fragment or a fraction of a cell that has been altered toexpress a Vip3 interacting polypeptide or fragment. Further, the Vip3interacting polypeptide can be the entire protein or a fragment thereof(e.g., a biologically active fragment) of the protein. It will beapparent to one of ordinary skill in the art that so long as the Vip3interacting polypeptide or fragment can be assayed for ligand binding,e.g., by a shift in molecular weight or activity, the present assay canbe used.

The method used to identify whether an agent binds to a Vip3 interactingpolypeptide can be based on the nature of the Vip3 interactingpolypeptide used. For example, a gel retardation assay can be used todetermine whether an agent binds to a Vip3 interacting polypeptide or afragment thereof. Alternatively, immunodetection and biochiptechnologies can be adopted for use with the Vip3 interactingpolypeptide. A skilled artisan can readily employ numerous art-knowntechniques for determining whether a particular agent binds to a Vip3interacting polypeptide.

Agents and cellular components can be further, or alternatively, testedfor the ability to block the binding of a Vip3 toxin to a Vip3interacting polypeptide. Alternatively, antibodies to the Vip3-toxinbinding site or other agents that bind to the Vip3-toxin binding site onthe Vip3 interacting polypeptide can be used in place of the Vip3 toxin.

Agents and cellular components can be further tested for the ability tomodulate the activity of a Vip3 interacting polypeptide using acell-free assay system or a cellular assay system. As the relevantactivities of the Vip3 interacting polypeptide(s) become more defined,functional assays based on the identified activity can be employed.

As used herein, an agent is said to agonize Vip3 interacting activitywhen the agent causes an insecticidal or cytotoxic effect, e.g., thecells may themselves exhibit one of the indices of cell death, such asreduced thymidine uptake, slower increase in optical density of theculture, reduced exclusion of vital dyes (e.g., trypan blue), increasedrelease of viability markers such as chromium and rubidium, and thelike.

In certain embodiments of cell-based assays, the cells are contactedwith the candidate toxic agent and the cytotoxic effect on metabolism ormorphology is noted in the presence and absence of the candidate. Thedifferential response between the toxin-treated cells and the cellsabsent the toxin is then noted. The strength of the toxin can beassessed by noting the strength of the response. The assay may beconducted directly as described above or competitively with knowntoxins. For example, one approach might be to measure the diminution inbinding of labeled Vip3 toxin in the presence absence of the toxic agentcandidate.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences of the Vip3 interacting polypeptide or Vip3toxin. An example of randomly selected agents is the use of a chemicallibrary or a peptide combinatorial library, or a growth broth of anorganism or plant extract. As used herein, an agent is said to berationally selected or designed when the agent is chosen on a nonrandombasis that takes into account the sequence of the target site and/or itsconformation in connection with the agent's action. Agents can berationally selected or rationally designed by utilizing the peptidesequences that make up a Vip3 interacting polypeptide and Vip3 toxin.For example, a rationally selected peptide agent can be a peptide whoseamino acid sequence is identical to a fragment of a Vip3 interactingpolypeptide or Vip3. The agents tested in the methods of the presentinvention may include and not be limited to, peptides, small molecules,vitamin derivatives, or carbohydrates. A skilled artisan can readilyrecognize that there is no limit as to the structural nature of theagents used in the present screening method.

In addition to simply screening candidates, the screen can be used todevise modified forms of toxins which are more specific or less specificto particular classes of insects as desired. The ability to determinebinding affinity (Ka and Kd) dissociation and association rates, andcytotoxic effects of a candidate allows quick, accurate and reproduciblescreening techniques for a large number of toxins and other ligandsunder identical conditions. Such information will facilitate theselection of the most effective toxins and ligands obtained from anydesired host cell.

The following examples are intended solely to illustrate one or morepreferred embodiments of the invention and are not to be construed aslimiting the scope of the invention.

EXAMPLES Example 1. Identification of Vip3A Interacting Proteins from anSf9 Insect Cell Line

Sf9 cells (S. frugiperda) were cultured at 26° C. in Grace's Insect Cellmedium (Invitrogen) with 10% FBS (Sigma-Aldrich) and split into newmedium by trypsinisation every 4-5 days. Fitly 15 cm dishes of cellswere harvested at density of about 90% by scratching and collected bycentrifugation at 500×g for 5 min. Cell pellets were washed 3 times with50 ml of 1×PBS to remove trace amounts of FBS and medium components. Thecell pellets were frozen at −80° C. overnight and resuspended the nextday in 100 ml of extraction buffer, containing 0.5% NP40, 0.25 M NaCl,50 mM HEPES, pH 7.4, 2 mM EDTA, 10% glycerol, phosphatase inhibitor(Sigma) and protease inhibitors (Roche). The solution was incubatedovernight at 4° C. for extraction. The crude protein extract wasseparated from the unbroken cells and organelles by centrifugation at34000×g for 30 min. Extracted proteins passed through either thecontrol, non-bound agarose bead column or a Vip3A-bound agarose beadaffinity column, prepared by the conjugation of trypsinized Vip3A to theagarose beads using a cross-linker kit (Thermo Fisher Scientific). Thecolumns were washed extensively with extraction buffer and the bindingproteins were desorbed from the column sequentially with an acidicbuffer (100 mM glycine, pH2.2, 1M NaCl) and alkaline buffer (50 mM CAPS,pH 10; 1M NaCl). Proteins eluted by alkaline solution were analyzed onSDS-PAGE followed by silver staining (Thermo Fisher Scientific).Detected bands were cut out from the gel and analyzed by MS analysis.From this, ATP synthase α, ATP synthase β, and Hsc70 proteins from S.frugiperda (fall armyworm) (SEQ ID NO: 1, 2, and 3, respectively) wereidentified as interacting with trypsinized Vip3A.

Example 2. Detection of Interaction Between Vip3A and ATP Synthase byWestern Blot

Sf9 cells were cultured and crude protein was extracted and separated asdescribed in Example 1. Proteins were bound to control beads or Vip3Abeads as described in Example 1, Eluants and total crude proteinextracted from Sf9 cells were each separated by SDS-PAGE and transferredonto PVDF membrane followed by detection with a monoclonal antibodyagainst ATP synthase α (Mitoscience) that cross-reacts with fallarmyworm ATP synthase at a dilution of 1:2000. ATP synthase α from theSf9 cells was detected in the elution from the Vip3A affinity beads, butnot to the control beads. Another analysis was performed using totalproteins extracted from S2 (Drosophila melanogaster) cells transfectedwith either an empty plasmid or a plasmid expressing a flag-tagged ATPsynthase β from S. frugiperda. The total protein extract was passedthrough either a column composed of non-bound agarose beads ortrypsinized Vip3A bound to agarose beads. Binding proteins were elutedfrom the column using 2× SDS loading buffer. Following SDS-PAGE andtransfer onto PVDF membrane, a western blot was performed using a HRPconjugated anti-Flag antibody (Sigma). The flag-tagged ATP synthase wasdetected in the elution from the Vip3A affinity beads, but not thecontrol beads.

Example 3. Co-Immunoprecipitation of Vip3A from Insect Gut andIdentification of Prohibitin as a Vip3A Interacting Polypeptide

Gut material was isolated from Helicoverpa zea and Manduca sexta 2nd-4thin star larvae. Gut from each species was homogenized and sonicated at4° C. in lysis buffer (PBS, 0.5% Triton-X100, 1× Roche complete proteaseinhibitor), the insoluble fraction was pelleted and the supernatant (gutextract) was saved. Beads were prepared by incubating Vip3A polyclonalantibodies with protein-A agarose in PBS for 30 minutes, followed byrepeated washing of the beads with PBS. A portion of the Vip3A loadedbeads were used to preclear the insect gut extract. To preclear, theloaded beads were added to the extract and rocked for 30 minutes. Beadswere then pelleted and removed. For each insect, co-immunoprecipitate ofVip3A was performed as follows: Precleared gut extract was dividedequally into 3 parts. Vip3A (full length) was added to one sample,trypsin activated Vip3A was added to the second sample, and nothing wasadded to the third sample. After rocking at room temperature for 60minutes, an equal amount of Vip3A polyclonal antibodies bound toprotein-A agarose (prepared as above for preclearing) was added to eachsample and rocked at room temperature for 30 minutes. Beads werepelleted and the extract removed. The beads were washed multiple timesin PBS, liquid was removed and beads were heated to 95° C. in SDS-PAGEloading buffer. The samples were then separated using SDS-PAGE and thegels were Coomassie stained. Bands that were unique to the Vip3Acontaining samples were excised for identification, and thecorresponding regions in the control sample were also excised asnegative controls.

Gel slices excised for ID were cut into 1 mm cubes. Pieces were washedwith 100 mM NH₄HCO₃ buffer and dehydrated with acetonitrile (ACN, Merck)two times. Samples were then rehydrated with 100 mM NH₄NCO₃ buffercontaining 10 mM DTT and then treated with 50 mM iodoacetamide in 100 mMNH₄HCO₃. Dehydration and rehydration were repeated two more times, andthen dehydrated gel pieces were rehydrated with 50 mM NH₄HCO₃ containing20 ng/μl trypsin (sequencing grade; Promega) and incubated for 12-16hours at room temperature. Peptides were extracted with extractionbuffer containing 50% ACN and 5% formic acid. The solution containingthe extracted peptides was evaporated to less than half the volume in avacuum centrifuge. Extracted peptides were separated by reverse phase ona capillary C18 picofrit 10 cm, 75 μm ID proteopep II column (NewObjective) in 0.1% formic acid using acetonitrile as an eluent at 400nl/min. Peptides were eluted on a nanospray source at 1.8-2.0 kV into aLCQ Deca XP-MAX ion trap mass spectrometer. Eluting peptides wererecorded in positive ion mode and automatically subject to CIDfragmentation by MS/MS. Data was then searched using MASCOT softwareagainst the MSDB database to identify the proteins. In both H. zea andM. sexta experiments, peptides matching “prohibitin 2” from B. mori wereidentified in trypsin activated Vip3A coIPs. The polypeptide sequencesfor each were subsequently determined (SEQ ID NO: 4 and 5, respectively)prohibitin-2 from H. zea and M. sexta were also used in subsequentexperiments (SEQ NO: 6 and 7, respectively).

Example 4. Isolation of Vip3A Interacting Polypeptide Genes from InsectLarvae

To obtain clones of the identified Vip3A interacting polypeptides, thefollowing Vip3 sensitive insect species were used for isolation ofcandidate interacting genes: Bombyx mori (ATP synthase α, ATP synthaseβ) Spodoptera frugiperda (ATP synthase α, ATP synthase β, Hsc70),Helicoverpa zea (prohibitin-1, prohibitin-2), and Manduca sexta(prohibitin-1, prohibitin-2). mRNA from insect larvae gut material wasisolated using the Straight A mRNA kit (Novagen), and cDNAs wereproduced using the AccuScript High Fidelity First strand cDNA synthesiskit (Stratagem) according to manufacturer's instructions. Receptor geneswere isolated by PCR amplification of corresponding sequences from firststrand cDNAs. The primers for PCR amplifications of ATP synthase β, ATPsynthase β, prohibitin-1, and prohibitin-2 were designed based on knownsequences of corresponding genes from Bombyx mori. Compatiblerestriction sites were incorporated at the 5′ends of the primers tofacilitate further subcloning of receptor candidates into the insectcell line cloning vector pIZT/V5-His (Invitrogen). The nucleic acidswere subcloned into pIZT/V5-His in-frame with C-terminal V5-epitope and6×His tags.

Example 5. Construction of a Yeast Surface-Displayed Manduca sexta cDNALibrary

Gut material from the 3rd instar of Manduca sexta larvae was collectedby dissection and immediately frozen on dry ice. 250 mg of gut tissuewas used for isolation of mRNA using an mRNA isolation kit (Stratagene)and following the manufacturer's protocol. Nine micrograms of isolatedpoly A+ RNA were used as a starting material for synthesis of cDNA byusing the ZAP cDNA synthesis protocol (Stratagene). cDNA wasdirectionally cloned into the pYD1 yeast surface display vector(Invitrogen) via. EcoRI and XhoI sites and transformed into E. coliXL10-Gold ultracompetent cells (Stratagene). After library amplificationin E. coli, the mixed plasmid population (plasmid library) was isolated,transformed into the Saccharomyces cerevisae strain EBY100 (Invitrogen),and transformants were selected on minimal dextrose plates [0.67% yeastnitrogen base (YNB), 2% glucose, 0.01% leucine, 1.5% agar]. Greater than2×10⁵ transformants were pooled and harvested by resuspending the cellsin YNB-CAA-Glu (0.67% YNB, 0.5% casamino acids, 2% glucose), aliquoting,and storing at −80° C. after the addition of 15% glycerol.

Example 6. Isolation of the Vip3 Binding Partner Serpin from Manducasexta Yeast Surface-Displayed Library by Biopanning

300 μl of Manduca sexta expression library in yeast was inoculated into20 ml of YNB-CAA-Glu media and grown at 30° C. for several hours (to anOD₆₀₀ of ˜2). 5-10 ml of grown yeast culture was centrifuged andresuspended in 20 nil YNB-CAA-Gal (0.67% YNB, 0.5% casamino acids, 2%galactose) media. This culture was incubated at 20° C. for 24 hours toinduce expression and to display library proteins on surface of yeastcells. Cells from 0.5 ml of yeast culture were harvested bycentrifugation, resuspended in 1×PBS buffer supplemented with 0.1% BSAand 20 μg of trypsinised, biotinylated Vip1A (bVip3A/T) was added to thecell suspension. Cells were incubated with bVip3A/T for one hour at roomtemperature on a rotary wheel, before they were harvested, washed andresuspended in 0.5 ml of 1×PBS buffer. 50 μl of streptavidin-coatedmagnetic beads were added to the cell suspension and incubated on icewith occasional tube inversion for 10 minutes. A magnet was then appliedto capture the beads and supernatant was removed. The magnetic beadswith attached cells were cultured in 2 ml YNB-CAA-Glu media at 30° C.with shaking (300 rpm) for at least 2.5 hours to separate the capturedcells from magnetic beads. Culture grown from beads was plated on large(15 cm diameter) minimal dextrose plates (0.67% YNB, 2% glucose, 0.01%leucine, 1.5% agar) and incubated at 30° C. for two days. All yeastcolonies from plates were pooled into 1 ml of YNB-CAA-Glu media andstored at −80° C. with 20% glycerol. This material was subjected toanother selection cycle described above and the whole selection(“panning”) process was repeated 7 times. After the last selection, amixed plasmid population was isolated from yeast cells and transformedinto E. coli. plasmid DNA was isolated from individual E. coli coloniesand the pYD1 plasmid inserts isolated from at least 20 clones weresequenced. After seven rounds of selection, nine out of twenty clones(45%) carried the gene for serpin. This level of enrichment afterreiterative selection strongly indicates affinity of cell surfacedisplayed serpin for bVip3A/T. In a negative control experiment (noincubation with bVip3A/T step was performed) no enrichment for serpinwas observed (i.e., none out of twenty sequenced clones carried theserpin gene). The polypeptide sequence of serpin from M. sexta wasdetermined (SEQ ID NO: 8).

Example 7. Vip3A Interacting Polypeptides Localized to Cell Surface inCultured Insect Cells

The pIZT vectors described in Example 4 comprising coding sequences forVip3A interacting polypeptide candidates serpin, ATP synthase β, orHsc70 were each transfected into Sf9 cells using Fugene HD reagent(Roche). Cells were incubated for 12 hours in Grace's Insect Cell mediumwithout FBS and then for 48 hours in complete Grace's Insect Cellmedium. Cells were trypsinized and transferred to confocal microscopedishes and allowed to grow for another 24 hrs followed by fixation with4% paraformaldehyde for 20 minutes. The surfaces of the fixed cells werestained by a well-known non-permeabilization method with theFITC-conjugated anti-V5 antibody (Invitrogen) and mounted with anti-fadereagent with DAPI (Invitrogen). Fluorescent confocal microscope was usedto check the distribution of the expressed protein in the SP) cells. ATPsynthase β, serpin, and Hsc70 were each found to localize to the cellsurface of Sf9 cells. DAPI was found to correctly localize to thenucleus for all transfected cells.

Example 8. Vip3A Interacting Polypeptide Render Drosophila S2 CellsSensitive to Vip3A Treatment

Drosophila S2 cells were cultured at 26° C. with Schneider's insect cellmedium (Invitrogen) with 10% heat inactivated FBS (Sigma) insemi-suspension and subcultured every 3-5 days. Two hours before theexperiment, S2 cells were split into 6 well plates and serum medium withthe attached cells was removed and replaced with fresh Schneider'smedium with 10% FBS. Attached S2 cells were transfected with pIZTvectors (as described in example 4) comprising coding sequences forHsc70, serpin, and ATP synthase α and β from S. frugiperda, using FugeneHD (Roche) for 12 hours. Additionally, S2 cells were co-transfectedusing similar methods with pIZT vectors expressing Hsc70 and ATPsynthase α and β or Hsc70 and Serpin. All transfected or co-transfectedcells were cultured for 48 hours. Then they were suspended and moved toconfocal dishes followed by the treatment with 0 or 200 nM of activetrypsinized Vip3A for 16 hours. Finally, cells were examined formorphological changes using fluorescent confocal microscopy. Results areshown below in Table 1:

TABLE 1 Vip3A interacting polypeptide confer cytotoxicity to DrosophilaS2 cells treated with 200 nM Vip3A ATP ATP synthase empty synthase α andβ serpin pIZT Hsc70 α and β and Hsc70 serpin and Hsc70  0 nM no no no nono no Vip3A lysis lysis lysis lysis lysis lysis 200 nM no no lysis lysisno lysis Vip3A lysis lysis lysis

Cells transfected with ATP synthase α and β, with ATP synthase α and βand Hsc70, and with serpin and Hsc70, were observed to lyse in thepresence of Vip3A.

Example 9: Detection of Interaction Between Vip3A and Vip3A InteractingPolypeptide by Far-Western Blot

The pIZT vectors described in Example 4 comprising coding sequences forprohibitin-1 and prohibitin-2 from H. zea, ATP synthase α and β from S.frugiperda, or Hsc70 were each transfected into Sf9 cells using FugeneHD reagent (Roche) for 12 hr in Schneider's medium without FBS andcontinued to grow for 48 hours in complete Schneider's medium with 10%FBS and appropriate antibiotic selection. Sixty hours aftertransfection, the cells were collected by centrifugation, lysed withdetergent buffer, and the proteins were separated from the remainingcellular extracts by centrifugation. Extracted proteins from thesupernatant were then incubated with Anti-V5 conjugated beads, and theV5 tagged Vip3 interacting candidates were partially purified andenriched by elution from the beads with SDS loading buffer. The proteinswere separated by SDS-PAGE and transferred onto PVDF membrane forFar-Western blot analysis. The membrane was incubated withbiotin-labeled Vip3A and Streptavidin-HRP followed by enhancedchemiluminescence (ECL) detection. Vip3A bound to ATP synthase α and βas well as Hsc70. A similar Far-Western blot analysis was performedusing biotin labeled Cry1Ab. The Vip3A interacting polypeptide were notobserved to bind with Cry1Ab, although the cadherin positive controlbound to Cry1Ab quite strongly.

Example 10: Interaction of Vip3A with Vip3A Interacting Polypeptides onInsect Cell Surface

The pIZT vectors described in Example 4 comprising coding sequences ofVip3A interacting polypeptides ATP synthase α from S. frugiperda, ATPsynthase β from S. frugiperda, or Hsc70 were each transfected into Sf9cells using Fugene HD reagent (Roche) for 12 hr in Grace's Insect Cellmedium without FBS and continued to grow for 48 hours in completeGrace's Insect Cell medium. The cells were then transferred to confocalmicroscope dishes and treated with 200 nM trypsinized Vip3A or V5-taggedCry1Ab for 30 minutes. Cells were then co-stained with Vip3A conjugatedto Fluor 546 (Invitrogen), and Anti-V5 Fluor 647 to visualize thelocation of the Vip3-interacting proteins using fluorescent confocalmicroscopy. Results indicate that ATP synthase and activated Vip3Aco-localize at the cell surface. However, ATP synthase and activatedCry1Ab do not. Additionally, ATP synthase and activated Vip3Aco-localize to the cell surface; however, ATP synthase and activatedCry1Ab do not.

Example 11: Dose Response of S2 Cells Transfected with Vip3A InteractingPolypeptides to Active Trypsinized Vip3A Treatment

Co-transfection of Drosophila S2 cells was performed as in Example 8using pIZT vectors expressing Hsc70, and both ATP synthase α and β fromS. frugiperda; Hsc70 and serpin, Hsc70 and both prohibitin-1 andprohibitin-2 from H. zea; Hsc70, cadherin from M. sexta, andaminopeptidase-N (APN) from M. sexta; or empty pIZT vector. Followingculturing for 48 hours, cells were suspended and transferred to confocaldishes, where they were treated with 0 to 800 nM of trypsinized Vip3Afor 16 hours. Finally, cells were examined for morphological changesusing fluorescent confocal microscopy. Cell death was determined by cellmorphology and propidium iodide staining. The percentage of dead cellswas calculated from each co-transfection and is presented in Table 2.

TABLE 2 S2 cell death in response to Vip3 and Vip3 binding partners % ofdead cells Hsc70 + Hsc70 + ATP Hsc70 + Hsc70 + cadherin + Empty Vip3A(nM) synthases serpin prohibitins APN Vector 0 3.5 1.5 2.5 4 4 25 18 6 83 6 50 37 17 19 7 7 100 64 34 33 6 6 200 92 64 56 9 9 400 98 81 87 10 7800 98.5 96 97 8 8

As expected, cell death is minimal in the samples transfected with Hsc70and cadherin and APN, the latter two of which interact with Cry1Ab, inthe samples transfected with empty vector, or in samples treated with 0nM trypsinized Vip3A. Cells co-transfected with Vip3 binding partnersshowed increasing cell death in the presence of increasing amounts ofVip3A, suggesting the Vip3 interacting proteins play a role in celldeath in the presence of Vip3.

Example 12: Knock Down of ATP Synthase and Hsc70 in Sf9 Cells ReducesVip3A Toxicity

Sf9 cells were maintained in Grace's Insect Medium (Invitrogen) plus 10%FBS and split into 48 well plates the day before the transfection at 85%confluence. Cells were transfected with 0.5 μg of dsRNA targeting HSC70or ATP synthase α and β (SEQ ID NO: 9, 10, and 11, respectively) or GFP(as a negative control) by applying 2.5 μl of TransMessenger reagent(Qiagen) in plain Grace's Insect Medium for 5 hr and replaced thetransfection complex with complete Grace's Insect Medium plus 10% FBSfor 48 hours according to manufacturer's instructions. Cells were thentreated with 200 nM trypsinized Vip3A in plain Grace's Insect Mediumwithout FBS for 48 hours after transfection. Cell viability was measuredby adding 50 μl of Cell-Titer Glow solution (Promega) and recording theluminescence with a Luminometer (Beckman). Results are shown in Table 3,expressed as a percent reduction of cell viability.

TABLE 3 Vip3 toxicity in Sf9 cells following silencing of Vip3interacting polypeptides ATP synthase ATP synthase ATP synthase Hsc70 βα α + β GFP 17.074 22.654 0.990 0.986 30.055

As shown in Table 2, the toxicity of Vip3 in Sf9 cells with Hsc70 or oneor both of ATP synthases α and β is significantly reduced compared tothe GFP control, where none of the Vip3 interacting proteins have theirexpression silenced.

Example 13: RNAi-Based Silencing of Hsc70, ATP Synthase α, and ATPSynthase β of S. frugiperda Reduces Vip3A Toxicity

Fall armyworm (Spodoptera frugiperda) eggs were purchased from FrenchAgricultural Research. In, (Lamberton, Min.), Eggs were hatched at 28°C. The neonate larvae were employed as testing insects. Insects weremaintained on a standard artificial diet supplemented with Cefotaxme andSpectinomycin at 0.625 mg/ml and 0.375 mg/ml, respectively. dsRNA toHsc70 and ATP synthase α or β (SEQ ID NO: 9, 10, and respectively) wassuspended in water and overlaid onto the surface of artificial diet ineach well of a 128-well plate at 100 ng/cm². Individual FAW larvae wereadded onto the diet of each well, for a total sample size of 128 insectlarvae. Larvae were transferred after three days into individual wellsof 128-well plates containing the commercial diet “Multiple Species”from Southland Product Inc. (Lake Village, Ark.), supplemented withCefotaxme and Spectinomycin at 0.625 mg/ml and 0.375 mg/ml,respectively, and overlaid with Vip3A protein at 100 ng/cm². Mortalitywas observed using microscopy and recorded daily. A summary of theresults is shown in Table 4.

TABLE 4 The susceptibility of RNAi-pretreated FAW larvae to Vip3A toxinon artificial diet supplemented with Vip3A at 100 ng/cm² Mortality (%)dsRNA for Target Gene Day 2 Day 6 Day 10 Hsc70 0.00 0.00 6.25 ATPsynthase α 12.50 12.50 37.50 ATP synthase β 0.00 15.38 53.85 ZsGreen7.16 37.50 56.25 (negative control)

Example 14: Pull-Down Studies

BT strain (C0548) from China collection harbors a variety of Bt proteinsincluding both Cry proteins and Vip proteins, totally about 20insecticidal proteins based on the genetic sequence analysis. C0548 wascultured in T3 medium for 5 days. The bacteria were separated from thesupernatant by centrifugation. Bacterial pellets were suspended andprocessed with Microfluidics, Western blot analysis confirmed that atleast Vip3A and Cry II are expressed in the Bt strain and weresuccessfully extracted.

Purified Vip3A interactors, e.g. ATP synthase (α and/or ß), HSC 70,prohibitin (prohibitin-1 and/or prohibitin-2) and/or serpin areimmobilized onto agarose beads and used to pull down Vip3A and othernon-Vip3A proteins. In one study, prohibitin proteins (prohibitin-1 andprohibitin-1) were covalently linked to agarose beads. Empty (control)beads or beads conjugated with the prohibitin proteins were incubatedwith C0548 extract, followed by multiple washings. Proteins binding tothe beads were recovered by SDS-PAGE loading buffer. Western blotanalysis performed with anti-Vip3A antibody showed that the Vip3Aprotein was successfully pulled down by prohibitin (prohibitin-1 andprohibitin-2) beads. The samples were further analyzed by massspectrometry for the presence of other Bt proteins in the eluted sample.

Example 15: Method for Screening Insecticidal Proteins

Bacillus thuringiensis (Bt) strain C0576 and AB88 are cultured in T3medium for 5 days. The bacteria are separated from the supernatant bycentrifugation. The intracellular total proteins are extracted bysonication of the suspended bacterial pellet. The secreted fraction oftotal proteins in the supernatant of the culture is precipitated with(NH₄)₂SO₄ at 100% saturation.

Drosophila S2 cells are transfected with Hsc70, ATP synthase (e.g., ATPsynthase α and/or ß) and/or prohibitin-1 and/or prohibitin-2 in the pIZTexpression plasmid and cells are selected on Zeocin media for one month.Cell lines stably expressing V5-tagged Hsc70, ATP synthase (α and/or ß),and/or prohibitin (-1 and/or -2) are obtained and continuously culturedin S2 cells. Cultured stable cell lines are collected by centrifugationand extracted by detergent buffer. Extracted proteins from S2 cells aremixed with intracellular (or secreted) proteins from Bt strains. Vip3Ainteracting proteins are coimmunoprecipitated with Vip3A protein byagarose beads conjugated with anti-V5 antibody and eluted with glycinebuffer, pH2.2, Eluted samples are analyzed by silver staining andprotein bands identified by mass spectrometry.

Example 16: Cell Based Method for Screening Novel Insecticidal Proteinsand Compounds

Expression cassettes on the pIZT expression vector encoding Vip3Ainteracting polypeptides are transfected into an insect cell line (S2 orother insect cell lines). A negative control of the pIZT empty vector isalso transfected into the same insect cell line. Successfullytransfected insect cells are selected for on Zeocin media for one month.The isogenic stable cell lines are used to screen agents, includingproteins from biological extracts. Agents which provide a differentialresponse in the presence or absence of the Vip3A interactingpolypeptide(s) are identified. These agents decrease the viability ofinsect cells expressing Vip3A interacting peptide(s), but do notdecrease the viability of insect cells which express the empty pIZTvector.

Example 17: Identification of Polypeptides that Interact with Other Vip3Proteins

The worker of ordinary skill can identify additional Vip3 interactingpolypeptides using other members of the Vip3 family based on the studiesand guidance provided herein. Examples 1, 3, and 5 disclose illustrativemethods of identifying Vip3A interacting polypeptides; however, similarmethods may be used to identify any polypeptides that interact with anyVip3 (e.g., Vip3D or Vip3E). For example, in representative embodiments,such methods employ insect extracts and materials from a correspondingsusceptible insect. To illustrate, Vip3E interacting proteins may beidentified using Vip3E isolated protein and extracts from Ostrinianubilalis (European corn borer) following methods similar to those ofExamples 1, 3, and 5.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the list of the foregoingembodiments and the appended claims.

What is claimed is:
 1. An expression cassette comprising a promoteroperably linked to a heterologous polynucleotide sequence that encodes aVip3A-interacting protein from a Spodoptera frugiperda insect that issusceptible to a Vip3A insecticidal protein, wherein theVip3A-interacting protein is a heat shock cognate 70 (Hsc70) proteincomprising the amino acid sequence of SEQ ID NO:
 3. 2. The expressioncassette of claim 1, wherein the promoter is functional in an insectcell.
 3. The expression cassette of claim 2, wherein the insect cell isa Spodoptera frugiperda cell.
 4. The expression cassette of claim 3,wherein the Spodoptera frugiperda cell is an Sf9 cell.
 5. The expressioncassette of claim 2, wherein the insect cell is a Drosophilamelanogaster cell.
 6. The expression cassette of claim 5, wherein theDrosophila melanogaster cell is an S2 cell.
 7. A cultured insect cellcomprising the expression cassette of claim
 1. 8. The cultured insectcell of claim 7, wherein the cultured insect cell is a Spodopterafrugiperda cell.
 9. The cultured insect cell of claim 8, wherein theSpodoptera frugiperda cell is an Sf9 cell.
 10. The cultured insect cellof claim 7, wherein the cultured insect cell is a Drosophilamelanogaster cell.
 11. The cultured insect cell of claim 10, wherein theDrosophila melanogaster cell is an S2 cell.
 12. A cultured insect cellline comprising the cultured insect cell of claim
 7. 13. The culturedinsect cell line of claim 12, wherein the cultured insect cell line is aSpodoptera frugiperda Sf9 cell line.
 14. The cultured insect cell lineof claim 12, wherein the cultured insect cell line is a Drosophilamelanogaster S2 cell line.
 15. A method of producing a Vip3A-interactingprotein comprising culturing an insect cell comprising the expressioncassette of claim 1 to thereby produce the Vip3A-interacting protein,wherein the Vip3A-interacting protein is a Hsc70 protein.